USPTO Patents Application 09830669

Application No. 09/830,669

Reply to Office Action of January 31, 2006

REMARKS

Claims 86-104 and 106-1 18 remain pending. Favorable reconsideration is
respectfully requested.

The objections to Claim 106 and Claims 1 16-1 18 are believed to be obviated by the
amendment submitted above.

Claim 106 has been amended to delete the reference to strain 1-2025.

Claim 116 has been amended to recite "one of the unconventional amino acids present
in the culture medium of step (b) is represented by an amino acid of formula I having L
configuration."

Step a) of Claim 108 is a selection process according to Claim 86 by which cells are
selected for their ability to incorporate an unconventional amino acid, which restores the
functionality of a protein essential to growth. The unconventional amino corresponds to the
amino acid which is initially encoded by the target condon. The resulting cells are mutated in
the protein translation machinery (ex: ARNt-synthetase genes, see also page 7, paragraph 1),
more particularly in their editing domain.

In step b), the cells selected in step a) are further cultivated with unconventional
amino acids, in order to produce proteins comprising one of these unconventional amino
acids.

In the sense of the present invention, an unconventional amino acid is an amino acid
other than the amino acid, which should be normally incorporated at a given site with regard
to the translated nucleic acid sequence. See page 2, lines 31 to 39 of the present
specification.

It is not mentioned that the unconventional amino acids used in steps a) and b) are
necessarily the same. On the contrary, it is recited on page 10, lines 11 to 21 that the
unconventional amino acids may have specific additional properties. In example 6, for

11

Application No. 09/830,669

Reply to Office Action of January 3 1 , 2006

instance, the mutated cells missincorporating Valine in place of Cystein (obtained according
to the process of Claim 86 - Example 3) are cultivated in a culture medium containing the
non canonical amino acids L-aminobutyrate.

In fact, the unconventional amino acids used in Claims 1 1 6 to 1 1 8 are those referred
to in step b) of Claim 108, and not the one encoded by the target condon in step a).

In order to make this point clear, it has been specified in Claims 108 and 116 that the
unconventional amino, acid carried out in Claims 108 and 1 16, is actually the one present in
the culture medium of step b).

In view of the foregoing, withdrawal of the objections is respectfully requested.
The rejection of Claims 86-1 18 under 35 U.S.C. §112, first paragraph, is believed to
be obviated by the amendment submitted above. Claim 86 has been amended to specify a
bacterial or yeast cell, which the Examiner has indicated as allowable (see page 3 of the
Official Action dated January 31, 2006). Accordingly, withdrawal of this ground of rejection
is respectfully requested.

The rejection of Claims 103-105 and 107 under 35 U.S.C. §112, first paragraph, is
believed to be obviated by the amendment submitted above.

Concerning the matter of the reproducibility of the invention to any aminoacyl-tRNA
sj/nthetase, aminoacyl-tRNA synthetase genes are well known in the art, especially among
bacterial and yeast species. Thus, it would not be an undue burden for one skilled in the art
to adapt the teaching provided in the specification conceming valyl-tRNA synthetase (ValS)
to other aminoacyl-tRNA synthetase genes.

Furthermore, as reported in the enclosed review of Aminoacyl-tRNA synthetase
(Delarue, M., 1995, Current Opinion in Structural Biology^ 5:48-55), it is also well known
that, despite the different topologies found in Aminoacyl-tRNA synthetase, their active sites
(editing domain) and mechanisms of action are similar (see in particular the abstract),

12

Application No. 09/830,669

Reply to Office Action of January 31, 2006

In view of the foregoing, withdrawal of this ground of rejection is respectfully
requested.

Applicants submit that the present application is in condition for allowance. Early
notice to this effect is earnestly solicited.

Respectfully submitted,

OBLON, SPIVAK, McCLELLAND,
MAIER & NEUSTADT, P.C.
Norman F. Obion

^ , u James J/ KeVlv/ Ph.D.

Customer Number Inr^ .

Attome> ©f Record

22850 Registratfon No. 41,504

Tel: (703)413-3000
Fax: (703)413 -2220

(OSMMN 06/04)

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Aminoacyl-tRNA synthetases

Marc Delarue

Institut Pasteur, Paris, France

Detailed mechanisms for «Bch step of the taction caia!yr«i by both ebss
' f ""'ifL" 'T!'^^^^ «yniheu«B have been proposed on the basis
P rfy«alk>gjiiph.c dab. of amlnoacyl-tRNA synthetases in complex with th«ir
dmerent substrates. D«plte the very dlffetent topolpgles oF rii tvvo cfasscs,
there are Sinking artd unanddpated chemical srmilaHties between their acUvg
sites and proposed rrwschaniBma.

Current Opinion in Structural Biology 1995^ 5748-55

49

Introduction

The unmoacyUcRNA syntheca^ea <aaRSs) azc a fiimUy of
enzymes fi^z emure the proper attachment of an axnino '
acid to is cogruce iKNaJ ^eracixie the pool of charged
cRNAs chat aire cs&cncial Got mRNA caiukcion inco pro-
ceina. AaRSi catalyse cfae charging of tRNAs in a two-
Atcp rcacdofi. lirsc, ATP reacts iwidi the amino dcid co
foriDi sn anunoacyl^adcziylate <and dlphoaphate). Second,
d^e activated amino acid is transfemd to the tRI^^A to
form die aniinoacyl-tRNA and AMP, Until 1989, only
three strucnucs of aaRSi were Imown ^^rjVrRS Tl^l^
ccMetRS (3}. and the «cGlnRS-cRNAGln complex (41,
where bs and cc in supcrvcript stand fbr BacHlus tf^h-
tilii and Euhcfickia colh lespccdvely). They aU shaEc a
soruccuial dootain called the Rossmann fold. oHglnally
observed in dehydvogenaia, which we now know to
be a characteriAtic of die so-called class I aaRS &mily,
which COnuins tO members [5*], A m^jor bxeakthxou^
in our underscanding of aaRSs came fiom the structuzal
fierexminadan of SetRS [6] and AspR5 [7]; both of these
enzymes lack the Rosmann fold and represent; a second
type of aaPUSs (class II) » Simultaneously, lequence analysia
showed that all known aaRSs can be auigned Co one or
the other of these two dajscs ([8]; Table 1).

Much pEOgress has been made in die post year on
Structuzal and functional studies of adRSs. The new
structures of class 1 aaRSs include GlnRS'-cRNAChi
in comt^ex with ATP and Mg?"^ [9^] and
pRS in complex with crypcophanyl-adenylate [lO-"],
New class tl sctuceures include: Thirmus thrrm^phHus
SerRS (oSerRS) alone as a complex with

two analogues of the seryl-adenylate 112-] or with
tRNASr [13-1; «AspRS-tRNAA*p bound to ATP and
Mg2-i- ccAspRS (where the sc in ffupeiscrjpc stands
for Saccharvfffyus atwisiae) in complex widi an amino
acylaced iRNA [14**]; «A«pRS alone (15-] or widi
asparcyl-adcnyktc [16**], "PhcRS [17**], although at a

pieliminaty su^ of zefinemenc, provides another escam-
pic of a dais II aaRS stnactun: (Table 1).

In this revievy; i ijvill fine ficKus on die scructucal
detaili that have been xecendy observed in di£Eeient
adcnylace-aaRS Con^lexea (one in each class); it ii
likely that the enzyiiudc leacdon mechanliois that have
been proposed [9*»,14-] in thcie systems wlU hold for all
aaRSs^ 1 will also emphasize the extraotdinary similarity
of the actiire site of c£2erent enoymes and some a^eccs
of the amino add binding pockets. The use of diesc data
to redesign amino acid specificity will be discussed.

In the second part of the artidfi. I will briefly review
odier biochemical and biophysical data, which contimies
to accumulace rapidly^ aflowing interesting comparisons
with structuial data and helping co fin the gxp between
systems where structural information is available and sys-
tem! 'where such infbrmadon does not exist yet.

Adenylate and related complexes: structuraJ
results

Class II hRSs

The tecent completion of the tefinemcnt of the
»CAspRS-ciRJ>lAAsp complex in the ptesencc of ATP
[14**] allowed the visualization of a bent conforma-
tion for ATP, sbowiog the leaving pyrophosphate group
pointing away fiom the O-phosphaxc-^ibose line and ata-
biliacd by at least one MgZ-^ ion. Several crucial toiducs
were identified, all of which ate scricdy conserved in class
n aaRSs. Two acidic tcsidues were found to bind a Mg^'*'
involved in the binding of the P- and T-phosphatca of
ATP. The counccrpani of these residues arc found in all
class n oaRSs (Fig. 1).

Soaking the «AapRS-tRKAAip complex with ATP
and aqxarcic acid leads diiccdy co fbrmadon of the

Abbrcvlallon
mtnoacyMRNA synthetase.

<d Current Biology Lid PSSN 0959-44OX

mm

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©015/021

Quaiemafy
U irrvicturt

Quuemary

dy

Ab
Pro
Stf
Thr
Hli

Asp
Atn

(M)

(OJI

(02)
(0(31
(03)

(a2)
(a2)
(a2)

I26-1
[6,11 Mi- 13-1

(CI2IU)

Clu

ta)

<a}

cyi

fa2)

Mvl

hi

(a)

lie

(a)

-ftp

(oa)

ClOMincMlon oT all uRffs on tho bMit «r (he p'W^fK* of iha tv««
ftSBfuluni Mqucncc* (High kmsicS; m letur amlna add coda
uiAd) tor cUm t and th» BBqumta mollfe 1, 2 and 3 cUss ii. On
(Ke iiAjduni tevtl ihli cofi Mp cndi to the ptMnce of • RoMmann
fold panHd p-theet (»r cUm I «nd m amipviliel p^heet (br dau D
Co]. On 1Kb Cunatorul lc%«l, ■mlnourylMlon TnvK/iaWy occuit On The
rOH of adencMtne 76 of LRNA br cSais i ull5i, M ihe 90H for
cl*M U mRSi (cKGcpi for PheRS> which amJrkMcyloied on the 2 'OH;
in CyiRS and TyrHS AminoacylAtlon mfghi ocxur on either 0/ the OH
iroupftl Iht ^|M(*n*ry •tiuouf* On £, cd/0 ot ell tm»m eniymei li mlta
ihowfi; tl H generally well conse(v«d BCTOxa s|MCle« («ClyRS, tot>^vyf
li m dime*), Subireup) coneipOMLrtB to both lequancM alignmenii

side each d**i and phy»tco>chemlcil propeilies of the amino aad
have b««n drown.

aminoacykted cA^A in die cryscal. The anwQ add
binding sice could be idendficct vAth, a positively
chai^god rujdue (Az^&3) located 4t ics base nuking
on ioq pair with the addic group of the iufa&czatc
Ude chain. A suicdy conserved negACivdy charged
residue was found co be reapoDsible fiir maintain-
ing the amino group of the amino add in place
(Kb. 1).

lb observe experimcnoUy the aspartyl-adenyktc inter-
mediate (fixu step of die reaction), it U nece&sary to
soaJc crystab of the cntymc alone; dm was done with
"AdpRS [16**]. £bt which d»ti for die atmctuiv of the
enzyme gJone also exist [15*]. It appcaxt diac die amino
acid binding pocket is esaendaHy rigid, with the e»>endal
Aig463 held in place duough a netwoik of electrottadc
interactions with at least throe odier charged tide chains.
RcdeftSgn of d)e amino add binding pocket of AspRS
to make it accept lysine instead of aspartate would of
course involve the mutadon of Arg483 inco a ne^dv«ly

charged amino add (as is the cose for LyaftS» as shown
by sequence alignment). But diii is not enough, as this
negative chaiige woidd then be in a very un&vourabie
enviionmenc; in Set, the whole pocket has to be elec-
Cfostatically redesignad. This discussion is very close in
spirit to die acgumencs developed mone quandtadvcly by
Hwang and Warshel [18]. in the more general context
of procdn engineering. It will be intettscing to see in
the structure of LysR5 how nacutt solved dtis problem
[19-].

The stnicDucs of SciRS in complex with two analogues
of secylr^adeayUce [12«>J show a ma^ed similatlcy to
AspRS in the active site; if one svperimpOAei the CU
carbon coozdinaces of the two enzymes and then ap-
plies this trarufbrxnaEion to bring die a^arcyl-adenylace
huo the £ramewoik of SerRS, the cc^phoaphatc lies ac
exactly the same place as the one seen in the two ana-
logues of seryl-adenylate (Pig. 2). The adennie ring is
sUgJxdy shi&ed, but all the intenccions desoibed a^ve
for AspRS (except those concerning sidc-cfaain lecog-
nidon) stiS hold £br SerRS: in &ct, die residues dm are
class II invariant Be at the same position, even chou^ the
backbone and the rest of the struczcure can vary (Hg. 2).
There is some discrepancy concerning the location of die
magnesiuiD-binding ate, but this could be explained by
the filCC diat different stages of the reaction arc observed
and/or by the presence of mote than one MgZ-t-

The fint step in die charging of a tRNA is dxc fornia-
don of an aminoacyl-adenylatc: a simple mechaninn
of in-line attack on the ckt-^ phosphodiester bond of
tbe ATP by the carboxyl gioup of die amino acid
bas been proposed [14-]. The transfer of this acti-
vated aminoac)^donyUte spedes to the tRNApzobaUy
proceeds duou^ an attack by the 3'OH of the xibose
moeity of adenosine 76 of die cRNA, ftom which the
proton has been extracted by one of the fine OKygcos of
die o-phosphate of the adenylate. Such an attack, which
docs not involve any proton actractor fiom die protein
itself, is not uncommon in cnzymadc mechanisma: in-»
deed, a dmilar mechanism hu already been postulated
for BcoBy. asparute carbamoyl ttansfittasc and, mote
recendy, the »«21 protein [20].

The iRNA-binding mode of SerRS has also been elu-
ddatcd in die past year [1?~], providing detailed in-
fbrmation on the structure of the tRNA itself (wliich
contains an unusual extra loop) and ita intetacdoxu with
die two very long and flrxiMc helices at die N^-terminal
end of the protdn. Unforcunately, the acceptor end of
the tRNA appears to be disordered in the crystal of tbe
cotnpleac, but .its interaction wida the synthetase is ex-
pected to be qualitatively riirijh f (but di6[erent in detail)
to the one observed in the AspRS syvtctn [^21**], as
vealed by a structural alignment between die Or-caibon
coordinates of bodi SerRS and AspRS.

It will be uitcicsting co see if die mechanism pomjlated
in [14^] can also be applied to PheRS, which amino-
acylates thm 2*OH of tRNA (an exceptional ^turc for

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SO Pro>tdn^ucl«k tcM immcHara

i J i

INlBRACnONS

(b)

0«IB

MOTIF 2

Aipfts 211 aLDRTroiAHCTRniDt wunwi»*t>u»LBHsrv

MAGHfisiuM BtNpiNo srra MCmF s

0016/021
092/037

Fig. 1. AJndfng 0/ amInoAcyl-AcWTylM
br class II aaftSs. U) Th» inieractton*
Involvwl In th« binding of the aspifiyl.
aderty*a» In WAapRS [U-L superlm-
posad on th« InterMlens Involved (n
th« bl^^ilng ch« IrhTbltlng ar>dloe of
the lerirl-adonylate tn «ScrRS (IJ^J.Tbe
>«aMu«$ InvolH/Ad mm boxed, with ihv
reridue fmm n^spRS fih^ «ppermo«
•nd thir Urom MSorRj shown bel^wL
The nature of rhftir inieraofvi with the
•minoacyl-od^nylart l5 shown: 0<N) MC
^ICM9 hydnogen-bonding fo the main
ch-rn oxygen (rtllfo||«n); w (iupencrlpt)
Indlcaies ihai Intmalon Is m«diatod hy
a wttar f7)olecura. ff rnr^licaied, the side
ch»in of dis amlm^ ackl k mpna/entcd.
The mimheHng in the two dlffenknc pco-
iftini ii alto IndlcBiod (iut*crrpi) and cor-
responds to the alignment given below.
<b> AllgnownC 0/ Aspft$ >nd 5erR5 m-
quence*. For simpllclcy, this dlignmem b
rotrJcrttd to the region of motifa 2 ar»d 3
ind 10 d nowly tdenilfied conserved re-
gion, whkrh, at laaM In AspRS, (s Imott-
CDRd In Mga* binding; iha Ittue of the
presence of difTerem lon binding 5lte(s>
in ScrRS sirir ha* to be molved. The
residues IndlcaiBd fin bold) hav« their
Side chaina In the same ipatlal poiirion
In the two enzymes and ere diretily so-
perimpoaable, av^ though scrrw signif-
icanr deviations occur In the rv»t of the
backbone of ih« &ah^ site (e* ragion
364^66 In "SotRS, probably due to the
presence of a prollneX

a class n oaRS): its chfee-dimcnsional structute dettrmi-
nation h near complcdoa [17-}.

The reccady solved structure of TVpRS In complex vidth
tryptophanyl-adcnylate [lO-J reveals a pattern of in-
ccracaohs chat doscly miiches due of TViAS with its
diffeient substrates (tyiosyl-adenybtc or cyiosine 11,21),
There u an almost peifeci 1:3 functional conespon-
dencc between stnjctiJiaUy equivalent renduea in the
rwo sysccms, rvoi cbowgh the cyveraQ sequence idetw
taty between die two enaymes i$ very low (13% fbr
272 unmo acids). The adenylate and amino acid bind-
ing pockets are deady mperiniposablc between the two
structures. The trypcophan-binding pocket shows good
sceHc complementarity with the substrate side diain,
suggesting that it should be aincaablc to specificicy mod-
ification using the normal principlei of protein scructuie

formation, m this case, essentially by alteration of side-
ch^ pocking. Redesigning the amino acid specificicy of
TyiRS M, however, not a trivial matter [22],

The structure of the TtpKS enzyme also contains in-
formation that could lead eo 0 piausihle model of its
interaction widl its cognare tRNA chat Twnild be con-
sistent with the one previously proposed for lyiRS [23],
with the acceptor and anticDdon arms of the *ame tRMA
interacting with two diOerent njonomera. The andcodon
end of the cRNA would be undiscortcd in both TyrRS
and TVpRS, conrrwy 10 yvhat is observed in the ClnR2
cOTipleac [4J. It should be lecaUod, however, Aac the
«RNA-binding mode of MetRS has been hnked to the
one observed with the GhiKS system, esp^aUy because
of the presence of a so-called acceptor binding domain
[24]. which IS absent in both TyrKS and TVpRS. Also
there is a sixth stzand with a left-handed type of connec-
tion in both MecRS and GhiRS that is absent both in
TyrRS and TYpRS. and this might be zelaced co a dificr-

mism

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BREVETS ET INVENTIONS

* SANTARELLI

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Asp3Xi

the wHtkMcyi adenyiMe blndlr^ sfces of
<«) y*af9R5 and <b) 5erR$. describMl in
|12*«] and 116^* f«sfMalvely. For clar-
ity. Afg223 and Ar^zs^ ni^flnds oi tfM
a-^S4)lim in AspRS and SerRS, r«spec-
trvaly) ha^ b««n omitted. The two struc-
twM ha:v« bwi luparlmpoAvd iMtng
the ci-cftrtx>n coofvAnaies only, traasport^
(ne iHe Mtonylaitt wteh rha darlvAd irani-
formailDn afterwaidf.

ent cR>lA-binding mode (both GhiR^ and MctRS can
function as mpnomon, whcroaa lyrRjS and IVpRS ave
both fiinctioiial diinen).

Lose year, Feiona et a!. [9**] used crywaHognphic data
on the complex of GlnR^-tRNAC^ bound to Mii2+
and ATP to infer die location of cbe amino acid bind-
ing pocket and to build a detailed enzymatic mecKanism
for the two-step reaction cacalyxed hy clott I aaRS». The
striking conclusiou of their work is chat the second step
is done in vfuy much die same way as by the dau tl
aaiRSs^ only in a diSerem geometry: die euiaction of a
proton &om the 2'OH of adenosine 76 of tRNA 14 aho
done by a fiec mygen of the adenylate a-phosphate.
Pteiona at, 19^] make a number of valuable cojiupar-
iflOns between structural and funcdonal results available
ibr GlnR^, TyrRS ot MetRS. A number ofpoincs dut
appear to be thared with class II aaKSs could also be
bifihlighced; however, a word of caurion It needed here.
Even chough the chemical nature of the amin'o add
side chains hxvolved in the different mteractiom ija both
class 1 and class II aaRSs often appears to be com^iarable
(see below)^ their positions axe not strictly superin^oa-
ahle; indeed, if one superin^ses the adenine tir^Ks, the
aminoacybbon reaction appears to take place onloppo*
aitc sides [14*"].

The first common point k that the way the adenine ring
of ATP IS Tecognited in GlnRS is highly reminiscent of

what is setn in both SerKS and AspRS: the Nl and N6
atoms ate bound to the amxde nitrogen and the carbonyl
oxygen of the main chain of the some reaiduc, namely
Leu261 in GlnRS (compare with Fi|c 1). Alio, the adc^
nine ring rests upon the aliphadc pccdon of Arg260, in
very much the same way as in dau II aaRSs CAjcs53l
of ttAspR^. ActuaQy, Peiona ti aL [9^] point out that
diis might be £mctionaily in^sonaar u the side cbaiii of
ucguiine is highly flexible, this mlg^t aHonv without dif-
ficulty a iUgfc^ cearxangement of dbe adenine ring moiety
during the dificzcnt steps of the reaction.

Another intere«ting feature is that the ademne ting Ves
on Gly42, a stricdy conserved reaichie of the first nyna-
turc sequence of dass 1 aaRSs; any other amino acid side
chain would interfere v/itti the binding of ATP. Odier
scricdy conserved residues of cither the first (M2s43) or
the second (Lys270) sigrxature aequ^nce of dan I aaRSs
are implicated in the scahHizadon of die transition state
of the reaction^ which inyohes a pcntovalendy bonded
phosphorus. This is to be related with eaiiier work u»-
ing kinetic measuremems^by Fersht a at. (reviewed in
[9*«]). Lys270 can be compared to Axg223 of motif 2 in
"'A^tRS in its role in binding the Or^hosphate.

Th e Ot^amino group of the amino acid appears to be sta-
bilized by an ion pair with an aspax&tc, which is con^
served in all (except lytRS) class I aaRSs^ at the end of

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52 ProtelrwHidelc acid tnlemctlom

^soand C; agaixiy this ifl cooipanble wich whac is i«en
for class II oaRSs.

The bindiz2g pocket for ^utamine in GlnRS contains
Ai^30, '7Vx211 and Gln255 at its ba^e, the g^taxnine
being ready to hydiOgcn bond co cfafi subscnCc Gln^5 is
held in place by a ncCwork of intcracdonfl 'wichin a pohr
cnvitonmcnt involving ihc chaiged side chain of Clu257
and the niain chain cavbonyl atom of Phe233. This sicua-
don is liighly xeminiscent of the bonding poc^ct^ of both
AspKS [16"I and lyrR^ p,22j. AU of chii has been
observed in the struccure of GlnR5 ficc of glutamine,
thereby implying chw the free cnisyme can bind ia sub.
urate without any significant rearrangement (no induced
tit). It is CO be hoped that the general natuxie of all the
obseTv;uion9 mentioned above will be confirmed by the
detailed 5Cr\kCtunl Kudieft on ttGluRS and »MctRS (S
Yolcoyanu, personal conununtcadon).

AaRS-tRNA recogrMtions modules and nodules

One o( the most useful concepts in ajialyxing aaRSs
of unkncywn $tructuie icmaim the concept of modu-
larity, originally introduced for Esdurichia mli AlaRjS
[25]. Apart from their catalydc domain. aaRS$ often
possess additional domains that help to make them more
specific in the tFJNA recognition proceu. Very often,
the»c domains can be idendfied thn^ugh flequence anal-
ysis (26*]. New sequences of uRS genes in distent or-
ganisms contimjc to appear but are too numetouti to be
eked in ihis review.

Struceuol data on che diOmnc domains found in aaR^s
have alAo appeared, and it is not inconccivahie that Tve
win «oon have a complete picture of the repettonc used
by diis &mily of enzymes. The structure of ™A^R$ (1 5- J
revealed an additional domain, of unknown function^
structurally similar to the histidinc-cozicaining phospho-
carrier protein HPr. The structure of "GluRS may teveal
a difien^ an dcod on-binding domain, compared -with
GlnKS. The scrucmra of «GlyRS 127*] and «HisRS
[28"], now appiOaching complete refinement (D Moras,
persona] conununicadon), will also provide the fint pic-
ture of a new andcodon-binding domain that should also
be present in ThrRS, It will be interesting to crompare
the forthcoming structure of «cLysRS [19'] with AspRS.
especially their amino acid binding pocketi. In addl-
uon, the xole of the N-tenninal exbensicn cpf mammalian
&aP^i i9 being acdveiy studied [29],

How these domains interact widi each other is sdU a
daunting c|ue$don. This question lelatea, of counc, to
the enzymes^ specificity and, in some cases, aBostery.
Some ptoigreaa has been made in the class II aaRS family
thrau^ a cateful structural analysis of the dimer intci£ice
[15*,30*] and throu^ pxobing by site-directed mutage-
neua [^O*], In the dau I &mily, some inovgmic sup-
pressors chat compensate Ibr impaired anunoacylation
in mutant] of GlnRS have been iaokeed and struc-

turally mapped to a region Hnldng the ca&Jytic and the
uuicodon-binding A^m^^^ [31*]-

The structure of che zinc-finger domain of MetRS has
also been solved recently by NMR methods [32*]. Thi«
is especially important bNccause zinc-finger domains have
been identified in many diffezenc doss I aaRSs by se«
quence analysia.

Meanwhile, experimencs designed to probe cRMA—
aaRS xnteracdons and to define the tRNA identity
I33'J are producing interesting new results. In some
ca*oB, it ptoved possible to change the specificity of
the xccognition [34J. The structure of the tRNA itself
and its tole in aaRS recognition continues CO be acdvtly
studied [35—37], The tolc of po^t-transcripdcmal modlH-
cadony of cRNA )n the rec<^;ninon process has also been
recendy ze-emphasized [38]. The part of the pmtcin xe»
sponsible &r recos;nition can in some cases be pinned
down to a few residues [39] and the vwicch to another
specificity can involve as litde as ckic methyi group [381,
hence the nodon of a nodule.

AaRSi may cepresent a unique system, as £ar as pro-
tein engineering is concerned, because the structure
of three different member? of thia lamily are (or are
about to be) known in both E. coli and X ihermophilus,
namely MetRS, SecRS and AspRS; the E. coli As-
pRS has also recently been solved (D Moras, peisonal
communication). Thus^ the study of aaRSs may rep-
resent one of the best opportunities to undexstand the
molecular basis Ibr thermophUidry. An hiteresdng ex-
perimenul approach has been underuken widi TyrRS
[40], whose structure ia known in the thermophilic or-
ganism B. steafpthennophUus. Chimeric versions of lytRS
were constructed using portions of sequence fiom the E,
coU and £. st^rathermt^hiha enzymes, and Che thermal
Stability of the resulting enzymes measured.

Other Mophyslcal and blodiemlcail studies

Althou^ the amount of inibrmadon given by these
siructunl results is impressive^ moxe data are snll needed
to refine our understanding of how erxxyuies of this
&mily fimcdon. As usual, le^ence data complement
refined structures weS because sequence consctvadon
profiles help to identlly fimcdonaDy important residues
[26*]. infi^rxnadon fiom the mulnple alignment of AlaRS
sequences^ secondary structure pzedicnons and site-di-
rected mutagenesis experiments has been used to identify
the position of motif 2 in AlaRS [41,42].

lb check hypotheses about the teacdon mechanism sug-
gested by crystal structures (which only give map<hf?tt
of some intermediate states of the Teacrion)* it is oftcm
iiuvucdve to generate single point mucano and meas-
ure their fimcdanal and kinedc properties [43-45^46*].
Chemical afi^nity labeling followed by miciosequencing
of the labelled pepddes [47] has also proved OSefid in
identifying residues in contact with che active t^tc, as have

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oehcr bJochcmic*] and biophysical mcMurcmentt such as
nuotescencc sCuciics [48].

One unreiohrcd issue that ia receiving more acteQijoa b
Che mechanism of proo&cading. It has been known for a
long time thac in tome inscances misActivBtioa can occur*
howcvet, upon tRNA binding, hydfolysis of the misac^
avaced aminQ add cakes place, so pjrevcnting the tRNA
ttgni hmg charged with che wrong amino acid Site-
directed mutagcncds in che infcmsd amino acid bindimr
pocket of MctRS [491 has been used co study thiTS-
5UC. but a stmctuial picture of ihe *hydtaiyth pathway'
for a mJsacclvaied amino add remaitis elusive* i=or ValR5
[50»], a mutant has been coginccicd widi de^rcued abil-
ity CO distinguish between the ri^c (valine) and tfac
wrong (isolcucinc) ainino acid. In any case, lyscematic
measuremencB of aH che Idnecic and binding properties
of an aaRS imino-acylacing (and charging) all po^le
«nino adds are certainly welcome [51,52-] and should
be made m the systems where detailed structunl infor-^
maclon is already available. This work in turn should
prova usefiiL in calibrating molficular dynamics simula-
Don methods ^splied to these systems [53],

Conclusion

The past year has seen the cluddicion of the structures
of several aaRSs (&um both class II and class I) with Uieir
diffi^nt jub»tr>te*. Striking and uoantidpated structural
and funcdonal similarities have been revealed, opening
the way for che ndona] design of mutane and of aaRSs
^th changed specifidty. This will probably demand
more sophisticated molecular modelling methods than
the already existing ones. In addition, structural data on
the same enzyme fiom bodi bacteria and eukaryoces (as
is available for AspRS) may prove useful for die design of
inhlbiiorj spedSc for prokftryotes. In this respect, an in-
tetcsdng experiment in bacteria has recently been done
[54*], Expression Of an intentionally inactivated tRJNA
synthetase, proved effective (and lethal) upon introduce
don of the ^nc encoding it into the genome of J5. {olt;
Jhh type of efiect had actually already bc^ observed for
lyrRS 155].

Data on a peculiar (and puzzling) feature of tRMA-
syndietascu* naznoly the antigenic nature chat Uiey some-
timea jhow, continue to accumulate. ValRS^ a class I
aaRS, has recently been identified as one of the major
antigenic decerminanu of Pit^modium vivax (V 5newln»
pcnonal communicadoD)* IleRS has also been impli-
cated in several autoimmune diseases, for instance in the
rheumatic disease myositis [56], Antibodies against dass
II aaRSs have been found as well (e.g, HisRS, GlyRS,
AlaRS and ThrRS in myositis and AsnRS in filoriosis
[6p. Therefore, our knowledge of aminoacyJ-cRNA
synthetases may eventually have medical appUcations.

Note added in pr<>of

te has reccnily been found that the dass n aaRS cacalytic
donuun fold is present in BixA, a biotin-prnteia lieise
that attaches biotin to various key mecaboHc proteins
mvotved m carbojcylation and decaibcayjation [57-], The
biotination reaction proceeds through the same type of
intermediate as found hi aaRS;*, namely an acyUdoxy^
w ^ expected tha t che same type of fold will be

observed m yot other proteins involved in metabolism
lor nisbuce acetyl CoA li^cs«

Adknowledgements

I Wish to thank P Ahari. H Bcdoudle. D Logan and t> Moras
fcr uMful Ccnmncnb on d>e knanuscripc. Sped«] dunks to CW
Career for eommiinicadne a copy of hk manuscript prior lo
publjeaboA. ^

Keferenceg and recommended rea ding

Papers of partJcujar Interc^ pubJiiherf wfihin ihe AA(\ua) pftrlod or
review. Save kmn highlighted as: -^-i p«rioo or

oT special fnccff^
«« of ountkndlng 1r

Bricfc P, Blow OM: Crrftol pfrwctutt: of a Mction wmtM, of
trwI-mNA Bxn^lwtaM cotI— »d %vWi finMlne. / A4oi Blot

Br|ek P, BJ-t TN, Blow DM; 54»ciuiv of irnxyLcmA tyn-

Brufila 5, Zch««r C RWcr JL: CryttaltonpfHC at

llM iafetfrMtao of ■MlMMtyi-tRNA >)nttnm, of

[5*^^f?*^ ■ — «~oyi-mrf/» J

£1 caH wtdi Am > Moi Biol 31(4411-424.

ftould^MA. P«roni JJ, Soil D, 5t*lU TA: CrMtel iOiMrtim oi Hm

f*JaCIii Co

d CD0 ^ilt4«lllllyUft^<A MdUuw-cthtj

1969^ 246:1135-IHa.

5.

Cflrty CW Ir. Cop%IUQm fi>ediafili«m and e^utlufiary rcla.
tkmAI^ to MrtMaeirMltNA fynlhttMS. Anna tttv Bkxhem
1993, aa:7)S-746. w o«fwm

tl^^ S"^*?*^*' aspects of hR5«, wilh a numbgr of illus^

ThSSS «»pec;aliy OA class l aminoaeyMHNA lyrw

6. Cusacle S, B«rTh»0»lomlnai C, Hartlain M, Nauar, Lttbttrmart
R: A fvvond cteu of iymbdaAe ftrydufv leveaM by X-r«y
aralyctf of e oofr slnictm of MryMKNA mMbrtme al 2-5 X

7. Ruff M, Krishnaswamy ^ Boeglln M, P©(ef5zman A, M^Mer
A. PwJiamy AO, f^ee* D, Thlwry JC Morq dan
II tadnfnc:yl.rtNA EyndieUfetl evyvtaJ fUtlctlW of yca«
?2?r^tf?'?^. •ymhetaM C0M|>lexcd wilh tUNAAir. S^nce

2$a:1 662-1609.

e. EnanI C, tXlarue M, Poch GanglofT I Mgr^S D: PartN^
«J amliwacyl-iaNA tyMhclaMi In |.n» dues on dta bub
Mr'JaS^*"'^"*^ s«|u«Ke inotiCi. NMlfW

9. P«rona JJ, RouM M\ Steicc tA: $ln>clinal batfg |br HtNA

•* T^^^5*^i5?! f IfciUmlnyUtMA tymhMaie.

Cftmi^^ 1993, 32;a7S6-^771.
Thh fnammoth paper is likslv to he th* rofof^nce «m dM»||«d mechanis.
tte and urvctural o/ cW \ toRSs for (|ulte tome llmo. Apart from

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a03e/037

54 Protegn-nucleic >dd fnt«ractSon9

iinicUinl rwuUf on \hm ar»RS-«RNACn In rompl^v with v«rioua
substratai, Ei combine a billfUoi vynrhesi* of all prevlousfy known stfi>c-
jiJrt» »oU kinetic miaturenwnu ^ dan » iiRSs and related compj^w
It C»n now bm updated ^sHth flO-]; mony of th« ItWas developed herv'
may Ond d COunlerpart in cbss II aaRSs (M [1 4*^>.

10- Dcjubtk 5, Bricogne G, Cllmiwe C Carter CW Jr. TWpia|A»l.
- tfcNA fynttMK cryntd ^tni^w ■vyoli » i!mpmS
P^-- 1 ^ tyrmyUKNA nnUMiM*. 5faic»u/v 1:17-32.

DCTCrtption of th« siArttur* of Another dass I aaRS, TrpRS, is a compttx
wirh rrrptnphanyl-Bdimylair. Th« paper afco descritM in d«4f| howr iwl
complej, oomw«» w»th rh« TyHW r«>utbi in 12). in addlclon. (i contairu
an *«T<«rt«in6 dr»cusston o<»ne^ »ppn»ch hi cryfliaJiogr^phic simctuiv
rwoJutw bM«d on a muclmom llfccMhood crllf^oo, ^ find th« phMt
of Krong r^netllora and COrfett the tnv^iope In th« Initial state of modd
tMJilding, ^

11. Fujlnaga M, BMhct-Colomtnas C, Yar^mchuk AD, Tukalo M.
• Cu«ckS: Btrfkltd CfTftoP itrtlClUf« MryUUINA milh.lw
STJt^IiML™*'''^ « rttolulloq. ; McW Bk^ 1993,

TOf pao«r contalni «n Intrnttlnfl dimiwion connmiitf ch* pottlbl*
ifloi^i#r bull of the theimopMltciiy of thh en<ym6. U also mha-
size* the nexJbWity of the iwo long cuhtlk» in tf4 N-tennfrwi pail of
tht Mqutfnca. ^

13.

Bdrhall Yaiwndiuk A, Mikm\o M, Lart«f) K, Benhac-GoloniU
fuu C UUrman B«J}«r B. Al^Mwrnn |. Cojbel C. Lrsrand
IP cr a/j CnnUl f«n>ct«re at UA rwluiiaii servUftNA

Th« fact Old th* two non-hydnoiyscable analogK bind at tho «a™ pjaco
■n (h« «nzym« piwldu ui^ng luppovt for th« hypoiheili ihai iW ac-
tualhr reflea the binding of n*Uve seryUdertylaic, 7h* nctwori^ in-
toractions b«t^«^ thv pr«icin and ihe «iibar»[M it c»f«rully Analyz«d
and dwcHM. Tha auihon .Uo dta ikm data at A ivsofutlon on a
complex bttvyeen ihc pioiein and ATP and Mni*^ which showi the ATP
fn a bM confornistion, «■ obwrwd 7n |>4**1.

13. BIpu V. YartfTXhuli A, TUkalo M, Cusacit 5: Thr 2.5 A Co*.

- tel Bindclw Atf ThefM^ I^MWpMW aaryUllNA ayndM^
conpkltad IBNA^. icrwice 1994, 263n404-Uia

This paper glvei •t/uctural detalli ol the Inraractiwis bcuvm SvrRS and
III c»|nai* iHNA. »Sow*nj ihM li h the «peciat featura of the rRNA,
namely the extra (or^g loop, and not the arttScocten, (h«i ii reoogntMd
by the protein. Untenunately, tha CCA end apptafs i» be diaofdctcd In
(he cryual. Paia hawe also been collected in (he presence of ihe ATPena-
log AMPPCP, which, cortrary to Ihe ATF, Is In an extended COn<om>atkM;
lh\9 H in accordance lesults al«0 datcrlfa^ In [14*-).

14. Cavatdli J, Ef^anl C. Re«s t^idS M, eoeslin M, Mitschier

- A. Mamn Cangloff Thltry |C, M«3f D: ifcT^SlS
sOe of yttat aBpartyMMNA ayntfieia^e: flmctml m! kmc-

13:127^337. ' '

a«s n coumerpait <rf I9"|. Jt describes for (he first Jme tha bindir» of
ATP to > cUm II aaRS, u vwlf «i a comploc of a class il aaftS wi|h art
amlnoacylaied cognata (Rna; all cfau li invanani imttdues i/r^lcated
in the aalve utu arv ld«ntined and a vnechanitm (i ptepoaed. S«v«raJ
miMarYtt have been conovucied and iheir Kinctiof^ prop>nles nwasuMi
to check ihls mechanlirn. These rtiuftj aie io be fupplernemed by ^
experimenCal dfUvrmlmOorv of aipaityl-adenylaiB bound Co "AuRS da-
scribed m Il«-J. See alio Ci2-,13*^. ^ oa-

15. ^•r^'ve M, Pot«r«man A, Nlbonov 5, Cartw Motm
♦ D, Thierry JC: CryrtBl itrMiire o4 % prokaryotfc anvtyl

tKNA a yn thalaw. £M3Q J 1^4, 14:9219-3229.
Dwcrtptfan of ih* stnictura of nAspHS; en addlikmal domalr^ ^ ptcv^
ai con^ared lo ihe yca« eiutyme, %^h pretents |he uma topology a»

prorvln HPr (of lha f, co/f |^kO«phoCranibnM mu^ Iraruport tySem)
thereby makinK this akructur* a tcxtbooif example of ihe modulariiv of
»«IJ5i» A detailed deicrifxion of the dimer imerfeea t» »l»o inckjdad, «
wall ai a pmpotal of a vtiucturat communleailM pathway befween the
catalylk and ihe arKlcodOn-MndiAg dbi^alng. See also (16**),

16. Polorazman A Del*n>» M, Thierry fC, Moras Z>, Synihesb
and tfe c ogmtfaii el aapartyUiacfybtg by Ihmm thmwwmMAikm
avpertyMRNA tyMlhctiM J Mol O/o/ 1994i 344:156-167^

Fi/5t 0irwclu«e of a clati 11 aaRS widt the cogruts (nailve) adanylste, which
in a aensQ fjcpn»cnt$ the »ltuatiori ato the Hrsi nep of the t*xtion has
been complatad. A comparlaon wldi tho i inicture of die enzyme aloiw

im n S*l) is alM gh^en, showing the rigid nature of iho amino DCid bind-
'"8 pockei.

w^pAai* te anbparallei of laMch only Iwd ai^

TTjWpaper mcrtbes the pnelrmlna'y chaio iracing of ^RS; the uf»-

SSStl?S ^^'^"^Mlh. largest 0^» .1^ ^^.^ rhS

typical (old of dau n hKSs, Le, an anilparel el B-sheer, even thouah •*-

^SLSS!!?^* Wbwnit b piObably not fUnaior«|, buc ha>

avDived from the umt attcestor u that of al) elaas II aaRSs. The aurhofS
t^S^ilSiT '^^'l^'^i^i!^ ^ o-*ubunrr for moHf 1, which
Jiliir!! 5 ^ eaHter Mudles. The ct.^»utmnli also comalns the fold
typical of dau IJ aaiiSs, The dimeriiatlon mode between (he a- and ^
■ubumt^simller to thca, d|fr«Hzaf'i>rt Interface observed If* both A^i^

16.

19.

Hwgny K War^ A: Wliy Ion |«r reverul by pmtain en-
^ncerlrtg b lafiObcIf <e auCceedL NMUit^ I96fl. 33^570-272.

One«( S. ThoecHtou Mr, Wtnura PL, Mirier AD, PUeau P

S!?SS*f;.*'^ '^•»*>*-'"^^ •yinh«is«e.;Ate/Aa;is94,

Aaji 123^1 25.

Cfy^Bli^ffmctlng to 2.1 A leaokrilon haw« been oUaJned kx coiHyfU
gene produo. The stnictuie of iha protein fs io appear

Scht^lns X Langcfi R, Warshel A: Why b^e muhagcnesfa itod-
iTsSTl'S^^e?" baia of raa p2lf NTMWe 5(rt«r 6/0/

21. Cavamlll J, H^ n fUjff M, Thiwrv |Q Mara» Oj Veast tRNA^
recogpUlon by li« cepiala daw II ambmcyMRKA aynihctaae,

Nahw 1993, 362:101-184, ^ jw«nc«»r.

The refined ilnicTum ai 2.9A ruoTutlon of ihli iRMA^aRS complex aU
to-js a mo*e detailed deicrtpdort of the interacdonc between the iRNA
snd tlic protein. The results ai* analyzed in light of odter biochemical
ej^o'i'nenta. The ganeraHzKlon of ih« lenjlb to other dan II eaR&t U
dbcuiMd (see also |13—J).

22.

De Prai Cay C, CX>cfcumh HW, F'enhc ARi Mftdrtk^Lm of the
ainlno add ipedAdly Of tyro^r^NA aynfheUae by preaehi

I. fBBS Lett 1?" '

23.

24.

25.

26.
a

1993, 318:167-171.
Bedouelle M, Cuez^ver^ V, Nagaotie DbcrtndMUion be*
^ »y»wrMRMA ey^theUse. Bhxhlmh 1993,

75:1099-1109.

S: SinMirel aiedl«MflM h itwanilnyi. »d indfalofiyi-UUSt^
syntheteA ui^^U a common overvn orientation of IRNA
bbidbig. fnx Nati Aad Sci USA 1991, 88:2903-2907.

Mrt hA, Regan 1^ ScKimmd P: Mcdukar irrwnttmuA at fkttM-
tional domabia alofil the leqiMnce ef elwtyMSNA Mihe«ve.
Nature 1963, 306:4Sl-447. -r"™«e-

petarue m, Mora« D: The amfamcyMRNA famllys medulea M
woib, SMicays 1993, 15:6^5-«6T,
A reeem review o/i mRSs> with wedal emphasb on die rockjlartty and
(niarpiay ba e »'e»i abuctgrat reaub and muhiple sequence •llgnw^s.

27. Logan DT, Cura y Ibuzel JP« Kem Moras D; CryrlalKzate
«l tfie g)ycyURNA ayntheiaae Inm r thtmofMJui and biHIel
oyttwejrvWc dbiU. y a«o/ arof 1994, 241.^^735.

Native and da rlva^ data on twQ oyital farm* have been obtained, An

tntaiprccabie map has bsen produced.

2a. Franddyn C Mairte D, Moras Dt Oymitbatfga of kbddyU

• «»NA aynOMlwe from £ «plL y A4o/ Btot 1994, 24i;273-^7
Crystals In two form9 hyve been obtained in the pn!5ence of hifti^ne
and ATP. The ralihsd struourt Should be available won.

29. Kovaleva NIkJtushnika T, Khselftv Lt Nudneide »lpW
phataae mtMfy aaaodeied ¥^ dta K^Milnai doMbTof
lvypiopK«ityl-iRi4A sypilbnwe* FEBS Ua 1993, 33S:i 96-202.

30. Eflani C, CawpneJll Martin F, Olrhdmer C, Morari D. CerMfoff

• I" itolc of dliMrEzatkM In yeast espaftyUwNA lymhittaic and
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The role of Ihe daq || Invariam Pr0273 In nwiif 1 was investJMied by
vte-dlrecied n^Ugene^il and analyzed In light of the Sfrudure of dM
fcAspRS-tRh4AA*l>coiTVl«>t.

mm

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ARELLI

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' ' 525!? iy-Jf^^ X Inokuch; H, Soli 0: nrndtaml eommunl.

»yntt>rt-r. fifTK N-£/ Aeti/ 5W 91:29t^/^
iKUaiiofi offlrujtanu ^/mrvi^d by »ice^ijr»cied mutogenesis Ibllo^ bv
Eene«i< Ml*ction tdrntiried two Emrion] |hu kiMnci wfih th» tnikfo
ri- Uih^ IRNA knd wkh mher. TWi piovkta cxp^Wmniil wl-

3*, ■'funriv D4retat Blan^uer S: MtlhimvMRNA synllB«Uw

1993, 231:1076-1069. r^«-»- / /vior pior

^li'lA^''^*^"^'!?^ '*!!^'* NMR-d«i<Vtg itaicturo Of thfl

• •nOMtti. JMof Bioi I99>, 334:257-260.

An UF«CMiaie JwvJewr of rRNA IdentUy In different iy«em$ by on« of the

34, Pygic^^M, Sgi, R, nor,na C: HfofUQy «wUchM

twwn tRNA* AmiiKwcyMnl bjr cUm I flutafninyi. mwA cImb ||
atpirtyMNA fy«iih«ca»cs. 6iocAe«Mj4<y 199*. M:99i2-»92i.
Lminw S, Hojmano HP, C3n Sprinat M: Thm S'Mntel

end (MCCA) of <4Na dtuwnino 111* «ifvcturf «iAd ciabnHy off
the amlMcyl Mxptor Hem. Proc Nftf/ Acarf Set USA 1993.
90:6199-6202. '

$l«intm SV, ICitHlBV LL; Mftcai c mm mtM far tftMA^toMe
rtaiypi fa p n , NucWe AcWl fef 1993, 3T:194l-194y.

HjjYm, WeahoT E, Cicgt An vmmol RNa lertUry
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WA, Froe /V*ff AC$d Sei USA 1993, 90:6776-4700

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41.

42.

45.

PMz J, FtDf*nEE C. BirK«lv Ctaa* A:

1;3 00-562,

N«ru/C Struct Biol 1994.

39- Schmln E, M«lnnftl T, Panufit JWiechglam Y, BlanqwM s-

TWO oddlc mkfciM of c cdr fMlli;o«»i^iRNA i^OkUm^ ad
M n«Btiv« tf««cfmJnaMtA toward* the fakwUm of nAiwMM*
tBMA anticodom, / Mai Biol 1993, 233:61 52»6.

Cuak-lvHitar V, Harm*nn M, Baldwin D, fi«doiMlk H: Mwolntf
tfie lUMOly ae«cfi-yMnla of baclcfM tyfoi>1^NA wpSZZ
S^^J!^?***^ "vwliitloriary apfiraachi y moI Btol 1993,
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D*vi$ MW, euechtar DD, Schiminei P; funcikmal dteeciion of
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Rjbai l>i FoupUna, Buaditxr DO, Divls MW^ ScHlmrnel P;
JdlagripliiE reptttsftfluiioii c<m«fvod domain of a daw
I? JJii^PtR?**^ unbMnwp, gtiuctw*^ Ptvnfn Sci 1993,
2:2259-2262.

P}rx EA, Pvnht AR: Invohmml of 'nM'234 Id cabdyiiv of lyi^

/iff)^ 1993, 32:13644^13650.

First EA^ Fmht AR: AAutHiim of Lyv233 to Ala Ifitroduco p<«.

1993. 32:13651-13657. ' ' ^

Flm EA. Fersht Aftj Muutkmal and hlMllc mlyab of a mofaib
tyrOiyl-IRNA lynihataM. Ptecftcmtoffy 1993, 32,13636-

47.

46.

S53?r-s?2?^ " iTtorf-rm&^y 1993,

A caraful study of the Idrwik steps oi pmtnoacytaCon usina kinoilc: mm*$*
wromentS in inut»nt» of Ty^ftS and Unrv fr«oin^ rol^WSpi.

BKmduo74 T, Roy S: rUlgresccficB ^»ectvo»CD(dc sivdv of

lyndtotaM. a#oc/iem^ 1993, »!926it9273. ^

Kfm MY, Choih C, Schulnun LM, Brunla 5, ^niboM^ki H:
Th« r^kraMp UfwMm fyndiHIe and adldng AvtcUora M

A«tf Sci t>i^ 1993. 90:11553-11357.

Schmidl £, 5chimmel P: Muttftiood bobtioa .f a Kim far edil.
1 ► ^ ^ iyn dia < aic, SeJItmc* 1994, 264:265-^67.

A muum of boh-udn^RNA ;iynlhMase wis consmjAcd lt«t lacle»
? ™^a' «bimy lo dl«Hnguish from tsotoudno. Ho»^*aver, tfw

vfcluo « comtdarably rMiucsd both for vallrw and Ipolwcinc
Incorporwlon. The motani nilf pos^sos the proper of hydrolyxifig Che
mbacxivaied ^Irio acid upon «NA binding. The authors imerprtai'helr
daia MM an Indlurion ihal rhm la • dtttlhcilve slevo for •
ftJftCtiorully indepcndenc from ihe amiiK» «cid bindWig il|a

51.

49.

50

Ir^i^'JSE?**^ n Cr.mw fi MvURNA »ymheai« from
yusl: OlscrtBiaBCiMi of BBivto adcfa by native phonhiw
rytattd fpedoa. iur S Bioehm 1992, 264:1 01 5-1 023;

52, Fi«<« W: Stonbach H, Cramw F: UronyURNA syntfitftM

of IRHATfcr. fla-^ Bfochmm 1994, 2301745-752.
Quantlficaiion of the binding ch»raoer|stic» of all posiibla imtno acids
to ihraonyURNA Aynlhda**.

53, Lau mc Kaiplus M. Mole;^ rccocnition in proickw; timola-
Hot* anolW* of Hibifral. bMb^ by ■ fymyt-lRNA »ndioCm
■tUam. } Mol Bioi 1994, 2^^046*1066. ^

54, Schmkit 5chlfnm«l P: f>oinlnaill iMhallry by apn^an of 0

• ^yacaAy hMctiwe cUii I \WA fyntheMfc: N^l Acod
Sci USA 1993, 90:6919-6923.

AliRnmor^-Sukiod rnMgpntatH wa» used lo genftrate a loxic *miun| of
iJoleuCirte-iRNA synthetase in E cafL Another mutalnn (double muunr)
diat WM al«o COnstrudad provod not to b« iMhal.

55. V^UCfos A, Bodouelte H: Rok Of RfSdoe ClulS2 bilhe db-
crimJnatiim b«tw««n WsntSmr BNAa by iynMyU»4A lyothviua
1^ sfeaAOCfttrmopMhit. y MOf 1992, 223:e6l-610.

56. Tayoff IN: A^to^lbodlet to andrMCyl-CRfMA •fiUhttMUf for
boluidne and ^ffthm: two addlUonal mtbctoM are anOmlc
bi Pvyodlb. J immwiOt I990, 144;1737>1743.

57. ArtymUik PJ, Rice DW, Polrrcqe AR, waiei P; A lale of two

* synlhcUsK. N^utc Struct Biol 1994, 1:756-760.

This artlcio Show« that blr^, a bioih^roieln ligase, and daw 11 jmlno
■cid IRNA synrhatuai krp smjohirilly homolo^ottf In their cataMic do-
mains*

M Ddarue, Unit* d*Iiniaunolagic Stmcomlc, Imeitut PaiMur, 25
njc Dr Roux, 75015 PaHs, F^ce.

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